Biochemistry 1991, 30, 8463-8470 Pearl, L. H., & Taylor, W. R. (1987) Nature (London) 329, 351-354. Polgar, L. (1987) FEBS Lett. 219, 1-4. Polgar, L. (1989) Mechanisms Of I"tease Action, PP 157-182, CRC Press, Inc., Boca Raton, FL. Rich, D. H. (1985) J. Med. Chem. 28,263-273. Rich, D. H., Green, J., Toth, M. V., Marshall, G. R., & Kent, S. B. H. (1990) J. Med. Chem. 33, 1285-1288. Richards, A. D., Phylip, L. H., Farmerie, W. G., Scarborough, P. E., Alvarez, A., Dunn, B. M., Hirel, P.-H., Konvalinka, J., Strop, P., Pavlickova, L., Kostka, V., & Kay, J. (1990) J. Biol. Chem. 265, 7733-7736. Schowen, K. B. J. (1978) in Transition State of Biochemical Processes (Gandour, R. D., & Schowen, R. L., Eds.) pp 225-279, Plenum Press, New York. Schowen, R. L. (1 977) in Isotope Effects in Enzyme-Catalyzed Reactions (Cleland, W. W., O'Leary, M. H., & Northrop, D. B., Eds.) pp 64-99, University Park Press, Baltimore, MD.
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Suguna, K., Padlan, E. A,, Smith, C. W., Carlson, W. D., & Davies, D. R. (1987) Proc. Natf. Acad. Sci. U.S.A. 84, 7009-7013. Swain, A. L., Miller, M. M., Green, J., Rich, D. H., Kent, S. B. H., & Wlodawer, A. (1990) Proc. Natf. Acad. Sci. U.S.A. 87. 8805-8809. Tang, J., Ed. (1977) Acid Proteases, Structure, Function, and Biology, pp 61-81, Plenum Press, New York. Tomaszek, T. A,, Jr., Magaard, V. W., Bryan, H. G., Moore, M. L., & Meek, T. D. (1990) Biochem. Biophys. Res. Commun. 168, 274-280. Venkatasubban, K. S., & Schowen, R. L. (1985) CRC Crit. Rev. Biochem. 17, 1-41. Viola, R. E. (1984) Arch. Biochem. Biophys. 228,415-424. Wlodawer, A., Miller, M., Jaskolski, M., Sathyanarayana, B. K., Baldwin, E., Weber, I. T., Selk, L. M., Clawson, L., Schneider, J., & Kent, S . B. H. (1989) Science 245, 6 16-621.
Catalytic Enhancement of Human Carbonic Anhydrase I11 by Replacement of Phenylalanine- 198 with Leucine7 Philip
V. LoGrasso,* Chingkuang Tu,* David A. Jewel1,S George C. Wynns,* Philip J. Laipis,%and
David N. Silverman*i* Department of Pharmacology and Therapeutics and Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, Florida 32610 Received March 13, 1991; Revised Manuscript Received May 17, I991
Carbonic anhydrase 111, a cytosolic enzyme found predominantly in skeletal muscle, has a turnover rate for C02hydration 500-fold lower and a KI for inhibition by acetazolamide 700-fold higher (at pH 7.2) than those of red cell carbonic anhydrase 11. Mutants of human carbonic anhydrase I11 were made by replacing three residues near the active site with amino acids known to be at the corresponding positions His, Arg-67 Asn, and Phe-198 Leu). Catalytic properties were measured in isozyme I1 (Lys-64 by stopped-flow spectrophotometry and l80exchange between C 0 2 and water using mass spectrometry. The triple mutant of isozyme I11 had a turnover rate for C 0 2 hydration 500-fold higher than wild-type carbonic anhydrase 111. The binding constants, KI, for sulfonamide inhibitors of the mutants containing Leu-198 were comparable to those of carbonic anhydrase 11. The mutations at residues 64, 67, and 198 were catalytically independent; the lowered energy barrier for the triple mutant was the sum of the energy changes for each of the single mutants. Moreover, the triple mutant of isozyme I11 catalyzed the hydrolysis of 4-nitrophenyl acetate with a specific activity and pH dependence similar to those of isozyme 11. Phe-198 is thus a major contributor to the low C02hydration activity, the weak binding of acetazolamide, and the low pK, of the zinc-bound water in carbonic anhydrase 111. Intramolecular proton transfer involving His-64 was necessary for maximal turnover.
ABSTRACT:
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C a r b o n i c anhydrase I11 is a major protein of skeletal muscle where it comprises as much as 20% of cytosolic protein (Gros & Dodgson, 1988). Carbonic anhydrase I1 is found in red cells and secretory tissues. These isozymes of carbonic anhydrase ~~
This work was supported by a grant from the National Institutes o f Health (GM 25154). Address correspondence to this author at the Department of Pharmacology and Therapeutics, Box 5-267, Health Center, University of Florida, Gainesville, FL 32610-0267. *Department of Pharmacology and Therapeutics. 1Department of Biochemistry and Molecular Biology.
0006-2960/91/0430-8463$02.50/0
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are useful for investigations of catalytic mechanisms using site-directed mutagenesis because they have large differences in activity with very similar backbone structures. Isozyme 111 of carbonic anhydrase has a steady-state turnover number, kat, for hydration of C 0 2 that is 500-fold smaller than that of isozyme 11, and the binding constant for inhibition by acetazolamide is much weaker for isozyme I11 compared with isozyme I1 (Sanyal et al., 1982; Tu et al., 1983; Kararli & Silverman, 1985; Engberg et al., 1985). Moreover, isozyme I11 has a pK, for the main activity-controlling group, zincbound water, that is lower by at least 1 pK, unit compared 0 1991 American Chemical Society
8464 Biochemistry, Vol. 30, No. 34, 1991
LoGrasso et al.
LYS6 '
I
FIGURE 1 : Stereodiagram of residues near the zinc (dotted sphere) in bovine carbonic anhydrase I11 from the crystal structure of A. E. Eriksson and A. Liljas (Eriksson, 1988). Water molecules are shown as individual dots, and the zinc-water bond is represented as a line connecting the two.
with that of isozyme I1 (Engberg & Lindskog, 1984; Kararli & Silverman, 1985). On the other hand, the backbone conformations of human carbonic anhydrase I1 (HCA 11)' and bovine CA 111 are quite similar; the root mean square difference in location of main chain atoms is 0.92 A, and is lower for many residues near the active site (Eriksson, 1988). There is a 58% amino acid identity between isozymes I1 and I11 (Tashian, 1989); among the major differences are three residues in the active-site cavity: Lys-64, Arg-67, and Phe-198 (their positions relative to the zinc are shown in Figure 1). Among the five isozymes of vertebrate carbonic anhydrase sequenced to date, these three residues are unique to isozyme 111. In HCA 11, these are His-64, Asn-67, and Leu-198. We have replaced these three residues in the active-site cavity of HCA I11 with the amino acids present at the corresponding positions of HCA 11. The replacements Lys-64 His and Arg-67 Asn in HCA 111, as reported previously, result in mutants with modest (3-fold or less) enhancement in k,,/K, for hydration of CO, (Jewell et al., 1991). Neither of these replacements at positions 64 and 67 causes the pK, for the zinc-bound water to increase into the pH range above 6 or to enhance the very weak catalytic hydrolysis of 4-nitrophenyl acetate. Histidine at position 64, which occurs naturally in HCA I1 and has been placed in HCA 111, is necessary for maximal turnover in the hydration of C 0 2 by providing a pathway for proton transfer between zinc-bound water and buffers in solution (Tu et al., 1989; Jewell et al., 1991). There have been no previous kinetic experiments that reflect on the specific function or role of Phe198 in catalysis by HCA 111. We have approached this problem using the site-specific mutant in which Phe-198 is replaced with Leu, the residue that appears in this position in HCA 11. In addition, the double and triple mutants with replacements also at positions 64 and 67 were studied to detect interactions between these positions in catalysis. Catalysis of the hydration of CO, was measured by stopped-flow spectrophotometry and the exchange of '*O between COz and water by mass spectrometry. The replacement Phe-198 Leu in HCA I11 caused major increases in kinetic constants for COz hydration and 4-nitrophenyl acetate hydrolysis and in the tightness of binding of some
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__ Abbreviations: HCA. HI, human carbonic anhydrase 111; Ches, 2-(N-cyclohexylamino)ethanesulfonicacid; Hcpes, 4-(2-hydroxyethyl)1 -piperazineethanesulfonicacid; Mes, 2-(N-morpholino)ethanesulfonic acid; Mops, 3-(N-morpholino)propanesulfonic acid; Taps, 3-[[tris(hydroxymethyl)methyl]amino]propanesulfonicacid. I
sulfonamide inhibitors. Thus, Phe-198 is a significant contributor to some of the unique properties of carbonic anhydrase 111. The catalytic pathway for the hydration of CO, is quite similar for isozymes I1 and I11 (Silverman & Lindskog, 1988). The substrate, CO,, reacts with the active site in which hydroxide is present as a ligand of the zinc. The subsequent release of product, HC03-, results in water bound to the metal:
CO, + EZnOH-
EZnHCOf EZnH20 + HC03- (1)
To regenerate the form of the enzyme active in hydration, a proton-transfer step follows in a separate and rate-contributing stage of the catalysis shown in eq 2 where B is buffer in EZnH20
+ B& EZnOH- + BH+ k-3
(2)
solution or water (Silverman & Lindskog, 1988). This is an essential step in the catalysis and requires that the proton transfer occurs at least as rapidly as the maximal turnover number. For isozyme 11, there are many studies to show that His-64 acts as a proton shuttle, enhancing activity by increasing the rate described in eq 2 (Steiner et al., 1975; Tu et al., 1989). This proton transfer is believed to proceed through at least two hydrogen-bonded water molecules situated between the zinc-bound water and the imidazole of the side chain of His-64 (Venkatasubban & Silverman, 1980). Wild-type HCA I11 has a lysine at this position which is not a significant proton shuttle at physiological pH, but data suggest that it does shuttle protons at pH >8 (Rowlett et al., 1991; Jewell et al., 1991). The low and pH-independent COz hydration activity of HCA 111 at pH 8 which approached 1 X IO' s-I (Jewell et al., 1991). The very small value for ester hydrolysis may not occur at the active site for C 0 2 hydration (Tu et al., 1986). 'Khalifah (1971) for C 0 2 hydration and Steiner et al. (1975) for 4-nitrophenyl acetate hydrolysis. Table 11: Inhibition Constants, Kl(Micromolar), Were Determined from R I ,the Catalyzed Rate of Interconversion of C 0 2 and HC03- at Chemical Equilibrium" ~~~
~
inhibitor HCA 111 F198L HCA 111 R67N-FI98L HCA 111 HCA I1 0.03 (0.007) 0.06 (0.02) 40 (0.4)b 0.6 (0.08) acetazolamide 0.004 (0.0007) 0.008 (0.003) 8 (0.08) 0.1 (0.02) ethoxzolamide OCN30 (IO) 30 (0.3) 50 (7) 30 (7) I30000 (6000) 30000 (9000) 30000 (300) 30000 (5000) "Measurements were made at pH 7.2 with conditions as described in the legend to Figure 2. No buffers were used. bValues in parentheses are the estimated, pH-independent values for K, describing the binding of inhibitors to the zinc-bound water form of the enzymes. These values were calculated as explained in the text using the values of pK, determined from the esterase activities (Table I). For HCA 111, these values were calculated by assuming a pK, of 5.2 for the zinc-bound water.
pared with the value of 3 X lo5 M-I s-l for wild-type HCA 111. There was no further enhancement in this parameter caused by the replacement Lys-64 His to form the triple mutant (Table I). Maximal values of k,,/Km for these and two additional mutants are given in Figure 4. Each of the mutants containing Leu-198 showed changes in k,,/K, consistent with an ionization of pK, near 7, assumed to be that of the zinc-bound water with similarities to HCA I1 (Table I; Khalifah, 1971). The presence of an ionization affecting catalysis was confirmed by the measurement of catalytic hydrolysis of 4nitrophenyl acetate. The resulting pH-rate profiles can be described as dependent on a single ionization with values of pK, between 6.4 and 6.9 (Table I). The catalytic rate of this hydrolysis by the wild-type bovine isozyme 111 is very small (the maximal value of k,,/K, is 11 M-' s-I; Tu et al., 1986). By this measure, the double and triple mutants of HCA I11 listed in Table I have activity nearly equivalent to that of isozyme 11. The steady-state turnover number k,, for hydration catalyzed by the single mutant F198L (Table I) did not vary in the pH range 6.5-8.5 and was approximately 10-fold higher than for the wild-type HCA 111 which is 2 X lo3 s-l in this pH range (Jewell et al., 1991). The double mutant R67NF198L HCA 111 had values of k,, similar to the single mutant, except at pH >8.5 for which k, increased with pH. This effect may be caused by proton transfer involving Lys-64. A similar effect was observed in the single mutant R67N HCA 111 (Jewell et al., 1991). There was further enhancement observed for the triple mutant for which k,, attained values comparable to those for HCA I1 of 1.4 X lo6 s-l (Khalifah, 1971). The proton-transfer-dependent rate of release of water from the active site, RH20/[E], was measured in the absence of buffers other than the substrate itself, CO, and its hydrated forms. RHp/[E] varied with pH for the triple mutant (Figure 2) in a manner similar t o HCA I1 (Tu & Silverman, 1985). The maximal value of RHlo/[E] for the triple mutant, 9 X 104 s-I, can be compared to the maximal value, near 5 X lo5 s-l,
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7-
ln
. E 0,
L K
I 6
I 8
7
9
PH
pH dependence of RH /[E] for (A) F198L HCA 111, (m) R67N-Fl98L HCA 111, and (.)264H-R67N-F198L HCA 111. The values for HCA I1 are represented by the solid line [see Tu and Silverman (1985)l. Solutions contained 100 mM total concentration of C 0 2 and HC03-, and no buffers were used. Experiments were carried out at 25 "C with the total ionic strength of the solution maintained at 0.2 M with NaZSO4. FIGURE 2:
for HCA 11. The values of RHlo/[E] for F198L and R67NF198L HCA I11 are similar, near lo4 s-l, and rather independent of pH (Figure 2). For wild-type HCA 111, the values of RHlo/[E] are independent of pH at 2 X lo3 s-I. Maximal values of RH20/[E] for additional mutants are given in Figure 5. Upon addition of the buffer imidazole, there was considerable enhancement of RHIO/[E] for the single and double mutants (Figure 3). Enhancement of RH2o by addition of imidazole is a characteristic of HCA 111 and the K64H and R67N single mutants (Tu et al., 1990) as well as HCA I1 and some mutants (Tu et al., 1989). This has been interpreted as evidence for proton transfer from the imidazolium cation to
Biochemistry, Vol. 30, No. 34, 1991 8461
Catalysis by Carbonic Anhydrase I11 E
1-13
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2
3 ~ 3m 1
tAaA.l,i
- 1 .s
1
kC8lhOl
Lys64 Arg67 PhrlS8
3 x 1DS
-0.7
M'S.'
Lys64 Am67 PhelS8 1 x 106
-0.6
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Dependence on the imidazole concentration of (filled ] (open symbols) R , / [ E ]catalyzed by (left) symbols) R H l o / [ E and F198L HCA 111, (middle) R67N-Fl98L HCA 111, and (right) K64H-R67N-F198L HCA 111. The pH was 7.2 at 25 OC with the total concentration of all species of C02at 100 mM. The total ionic strength of solution was maintained at 0.2 M by the addition of Na2S0,. FIGURE 3:
I
R67NF198L F198L HCA 111 HCA I11 HCA I11 HCA I1 1.03 & 0.05* 0.96 i 0.08 1.05 0.07 1.05 0.03'
*
(RI)H o/ (RILO RH10/RD20 2.4 & 0.4b 2.7 & 0.7 4.5 f 0.5 8.0 f 0.7' 'Data were obtained at an uncorrected pH meter reading of 7; other conditions were as described in the legend to Figure 2. bKararli and Silverman (1985).
